Manufacture of 17-alpha steroids by genus trichothecium



MANUFACTURE or 17-ALPHA STEROIDS BY? GENUS TRICHOTHECIUM j 1 Herbert C. Murray, Barry Township, Barry County, and

acids are: usually utilized, The 21-acetate is 'a practical example of a suitable ester.

- Morphology and. Taxonomy of Fungij pages 589 and Peter D. Meister, Kalamazoo Township, Kalamazoo V County,'.1\1ich.,'assignors to" The Upjohn Company, Kalamazoo, Mich., a corporation of Michigan No Drawing. Application August 9,1957

Serial No. 677,234 a 37 21 Claims. (Cl. 195-51) This invention relates to a process for the'fermentative production of oxygenated steroids. More particularly it Hyalodidymae of: the order 'Moniliales.

It is an object of this invention to provide a novel a method for the introduction of oxygen into asteroid molecule, thus producing valuable "compounds having 1 pharmaceutical activity and intermediates therefor. An-

s s s atu u i esml um t hydr xy ate a steroida Hydroxylation occurs chiefly; in; the: 175 position; however, the ll position may becomehydroxyl ated,. or: concomitantly; several oxygenatio ns may, also talce place; for example,.;the 1-land' ;17'-'positi h ah d yl tsd n si e st p-1 n e i n r, the t d ct n heh ox :s'rohp a e.

not limited as to type or number of?suhstitnents. v :The'

essential requisite for operativenessin thepresent process Other ob 6 15, Blakiston, Philadelphia, 1950, describes the closely related Cephalothecium as possessing conidia in the form ofheads whereas in Trichothecium the conidia are single at the tip of the, conidiophore. Both Cephalothecium and Trichothecium produce somewhat pear-shaped hyaline or pale pink spores that are two-celled, the basal cell being smaller than the terminal cell. The genera Cephalothecium and Trichotheciurn are often considered synonymous in theart, and as used in-the specification and claims the genus Trichothecium encompasses the genus Cephalothecium. The genus Trichothecium (Cephalothecium) is of the family Hyalodidymous Moniliaceae of the family Among the species of the genera useful in the oxygenation of steroids ar fi tio d Cephalothecium ros eum ATCC 8685, 1

Trichot he'cium arrhenopum, Trichoth'ecilm candidum,

Trichothecium roseum NRRL 1665, Irichoth eciuiizplasmoparae, and

Tr'ichorhecium polybrochum;

tures of; typical strains of Trichothecium (Cepha'lotheo ium useful in the practiceof this invention. The cul .f. g steroids operative in the methodfofithe pr esentfor;17ehydroxylationisthat the starting steroid must conhydrogen, replaceable 'or substitutable"group-ah v tached to the l7 -position, for example, atertiaryhydrogen atom attached to the 17-position carbon atom. S uch Compounds contain the nucleus:

1 through 16 inclusively, especially Ill-methyl, 13 -fmethyl and 1'0,13-c limethyl groups. Double bonds may be present and/ or other positions, or the double'bonds mayib'e substituted by the addition of halogen or hydrogen j halide. Particularly valuable starting steroids are those lia iiirl g 1 to "and including 22 carbon atoms infthe' 'arbon-to "carbon skeleton and more especially those 'havi r ketogroup; Where the"17-positionisunoxygn "d-o'i oxygenatabl e, hydroxylation at 17 is aCCOtIiQIiShdZZQHoW ever, an unoxygenated or oxygenatable ll posit'iorl is simi- Q tures icurrentlyjlisted,by each of the two repositories are for the sake of convenience.

American Type Culture Collection:

*seumyKJBJRaper, NRRL. Strain 1665 ent -LTrich0theciam cystosporium Duddington, Duddin g ton strain Trichothccium roseum Link, Cain strain. t Culture of the fungi for the purpose and practice of this: invention may be many medium, liquid or solid, 7

favorable to the growth of the fungi, in accordance with procedures recognized in the art or as shown in United States .,Patent 2,602,769.

7 used, liquid media are well adapted to use under aerobic While solid media may be layer'or the preferred aerobic submerged fermentation conditions. Suitably theme'dia should contain sources of available carbon, nitrogen and minerals altheugh o f course there can significant growth and development n sr sthan p mum d ion V Available earbon may becarbohydrates; starches,

ge nized starches, .dextrins, sugars, molasses" ofcane, beets q shumr uc se; fr ctos m nnos galacwse, i m tasai i r e l e qtr nt s s A in Pe -Y V ts vi qiei a sl s roh aqe ai s da a e; i 'as oqi lms r te lower fa y c d r fat y-Q" fatsare illustrative of othermaterials "1' ,assimilable carbon forthe energy and swath/"re u r m n s qf h n it M x re o a i us ble bl The biological oxygenationis achieved bya specie's of fungus of the genus Trichothec'ium. Bessey,"E. A.,

ephalothecitim roseum 8685, Ti'iehothecium 1 7 0-1 ra'albureau voor Schimmelcultures atBaarn, Hollandi' v I Trichoz hecium lu'teum Petch, Strain Boedijn and ee. rassu ilal le err im y be provided y .s'o'lw. f insoluble; vegm bl or: animal, protein; soybean r T 1; meal, lactalbumin, casein, egg albumin, whey, distillers V l j i peptides, amino acids, ammonium salts for example am-' monium tartrate or ammonium sulfate, nitrates for example sodium or ammonium nitrate, or acetamide. A

As mineral constituents the medium may contain, either concomitantly present or added, available calicum, cobalt, gallium, iron, magnesium, molybdenum, and potassium. Sulfur may be provided by sulfates, alkyl sulfonates, sulfides, free sulfur or cystine, thiamine or biotin. Phosphorus, particularly in a concentration at or about 0.001 to 0.07 and preferably 0.015 to 0.12 molar, may be present as ortho-, meta'-, or pyrophosphates, salts or esters, phytin, phytate, glycerophosphate, sodium nucleinate, or casein. times desirable.

Otheraccessory growth factors, vitamins, auxins and growth stimulants may be provided as needed or desired.

Carbohydrate and fat are not essential for the growth of Trichothecium but carbohydrate provides an economical energy source for the fungi. Amino acids or proteins, separately or together, are desirable for aiding completion of the life cycle of the fungi.

Suspension agents, mycelial carriers or emulsifying agents such as filter earths, filter aids, finely divided cellulose, wood chips, bentonite, calcium carbonate, magnesium carbonate, charcoal, activated carbon or other suspendable solid matter, methyl cellulose, carboxymethyl cellulose or alginates may be added to facilitate fermentation, aeration and filtration.

The selected species of fungus of the genus Trichothecium may be suitably grown in a medium containing available carbon, illustratively carbohydrate such as a sugar or starch; assimilable nitrogen, illustratively protein, and

Boron and iodine in traces are some I utilizable mineral constituents preferably including zinc,

Inoculation of the fungal growth-supporting medium.

with the selected fungus of the genus Trichothecium may be accomplished in any suitable manner. grow well at a temperature of about twenty degrees centigrade and growth of the fungi is readily promoted by maintaining an incubation temperature of about fifteen to thirty degrees centrigrade; however, lower or higher temperatures not greater than about fifty degrees centigrade are also operative.

The steroid may be added either before thermal or other sterilization of the medium, before or at the timeof inoculating the medium with Trichothecium, or later, for example, after a 24 to 48 hour growth period. The steroid to be oxygenated may be added at any desired concentration although-a concentration of more than. two grams of steroid per liter of medium, depending upon the steroid, retards but does not prevent mycelial development. may be accomplished in any suitable manner especially so as to promote a large surface of, contact of .steroid' substrate with the oxygenating activity of the fungus, 'such as by dispersing the steroid substrate, either alone, with fungal growth. It need be maintained only withinsuch range as supports life, active growth, or the enzyme activity of the fungus.

While any form of aerobic incubation is satisfactory for the growth of the selected fungus or fermentation of the steroid substrate, the efficiency of steroid fermentation is related to aeration. Therefore, aeration is usually controlled, as by agitation and/or blowing air through the fermentation medium. Aeration may be effected by surface culture or under submerged fermentation conditions. Aerobic conditions include not only the use of air to introduce oxygen, but also othersources or mixtures containing oxygen in free or liberatable form. A wide range of readily 'ascertainable rate of aeration is practicable. In using air as the aerating medium, a desirable rate of aeration is about four to twenty millimoles of oxygen per hour per liter as determined by the method of Cooper, Fernstrom and. Miller, Ind. Eng. Chem, 36, 504 (1944). Under some conditions it is desirable to utilize different rates of aeration during the fungus growing or developing stage asv contrasted with the steroid fermentation stage. Aeration is suitably modified by using superatmospheric or subatmospheric pressures, for example, thirty pounds per square inch or ten pounds per square inch absolute. Oxygen uptake may be facilitated by the presence in the medium of various catalyst such as ascorbic acid, glutamic acid, citric acid, lactic acid, tyrosine, or tryptophane.

The time required for the fermentation of the steroids varies somewhat with the procedure. When the steroid substrate. is present at the time of inoculation of the medium, periods of from eight to 72 hours may be used. However, when the steroid is added to the fungus, after substantialaerobic growth of the fungal organism, for example, after sixteen to 24 hours at optimum temperature, the conversion of steroid substrate begins immediately and high yields are. obtained in from one to 72 Microbiological contamination maybe re- Trichothecia 1 The addition of steroid substrate to be fermented a dispersing agent, or in solution in a water-miscible orhours, 24 hours being generally satisfactory. The steroids may be fermented in a simultaneous or sequential hetero} fermentative procedure resulting in other useful products, which are recoverable according to procedures known in the art. These auxiliary fermentation products may be separated from the fermentation beer either at the same time, before or after the fermentation is complete with respect to the steroid fermentationproducts. Illustratively, fermentationof steroid by means of Trichothecium roseum produces oxygenated steroid as well as antibiotics, the antibacterial rosein and the antifungal Trichothecium recoverable by extraction with chloroform or ether followed by chromatography.

After completion of the steroid fermentation, the resulting fermented steroid is recovered from the fermentation reaction mixture. An especially advantageous manner of recovering the fermented steroid involves extracts ing the fermentation reaction mixture, including the fermentation liquor and mycelia with a water-immiscible organic solvent for steroids, for example, methylene chloride, ethylene chloride, trichloroethylene, ether, amyl acetate, and the like.. The fermentation liquor and mycelia may be separated and then separatelyextracted with suitable solvents. The mycelia may be extracted with either water-miscible or water-immiscible solvents, acetone being efiective. The fermentation liquor, freed of mycelia, may be extracted with water-immiscible solvents. The extracts can be combined, either before or after washing with an alkaline solution illustratively sodium bicarbonate, suitably dried, as forexample over Alternatively, steroid fermenting enzymes of a growth The temperature during the period of. fermentation of 1 the steroid may be the same asthat found suitable for anhydrous sodium sulfate, and the purified fermented steroid'obtained lay-recrystallization from organic solvents or by chromatography.

The 17-hydroxysteroids producible by the process of this inventionare useful as chemicalintermediates as well advantage in retardingfungus growth and instab lizing limiting.

I 7 13 .ethy lene dichloride-acetone :1

emulsions where lipoph'ilic used.

of the present A medium having a composition of twelve grams of corn steep solids and ten grams of glucose diluted to one liter with tap water was adjusted toa pH'of 4.5 and sterilized by-heat." A twelve liter porter of this medium was inoculated with spores of Trichothecium roseum Link NRRL 1665 (synonymous with Cephalothecium roseum ATCC 8685) and grown under agitation and aeration at a' rate of 0.5liter perminute for a period of 48 hours. Then 4.8 grams of' 1 1-desoxycorticosterone ZI-acetate dissolved in 150 milliliters of acetone was added and the fermentation conditions were continued for 48 hours. At that time the beer had a pH of 5.85. Both the beer and mycelium were extracted four times, each timewith three liters of methylene chloride. The methylene chloride extracts were combined and washed twice, each time with one-tenth by volume portions of a two percent aqueous solution of sodium bicarbonate, and then twice with 'one-tenth by volume portions of water. The methylene chloride extract was dried with anhydrous sodium sulfate andthen concentrated to a small volume upon a steam bath to leave 7.65 grams of oily residuei The residue was redissolved in SOOfmilliliters of ethylene dichloride and chromatographed over'40'O grams of Florisil synthetic magnesium silicate"using'650-milli liter portions of eluting solvent asindicated'in 'lable "I.

Chromatographic analysis, Table 1 Fraction Solvent -E1uatSolids,

i Mill l l 0 ethyene dichloride-acetone 12:1

1 i i dn ethyene dichloride-acetone 3: 1,

i neutrino combined, dissolved in sixty milliliters ofethylene chloride and re'chromatog'raphed over 35 "grams" of Elorisil synthetic magnesium silicate, using as eluting-solvents 52325356 tagentsf have heretofore been The following examples. are .9 I

Eluate solids, fractions12 through 15inclusive, were x two portions of 35 milliliters each of the following 'solione"acetone fraction. Thefirst ethylene dichlorideacet onexl z l fraction eluted "118,. milligrams of i crystals primarilyconsisting of 26B, 17 5:, IZI-t'rihydroiiy nene-3,20-dione. Two recrystallizations -from methanol pregnene-3,20-dione having a melting point of 229 to 233 degrees centigrades The; infrared spectrum, confirmed the 7 structure.

vents 220-milliliter portionsof solvents in the following 1 order:. ethylene dichlorideyethylene dichloride-acetone 8:1 twice, ethylene dichloride-acetonei'rl twice, ethylene dichloride-acetone 3:1 three"times, ethylene 'dichloride.

- vents in their respective orderr'ethylene dichloride, ethylene.dichloridewacetone 12:1, 1051, 8:1',L5.":'1, 1:1, and

produced 26.5 milligrams of 618,17b2,21 ti'ihydroxy 4- {acetone 21 three times, ethylene dichloride-acetone 1:1,

.1 and f acetone twice: "The" second ethylene dichlorideacetone 1:1ieluate'was"evaporated to yield' 84.8 milligrams of an oil which was then crystallized from etheracetone.- The crystals were separated-by decantingand reerystalli zecl olnceifrom 0.5v milliliter of methanol' to ga milligrams of crystals of llu, l7q,2l-trihy- .droxyl-pregnene 3,20 dione having amelting point of centigrade and characteristic identifycosterone Zl -acetat e, hYQQKYPIQ w Q e 7 ExAMPLn3 7 Following theprocedure in Example 1, using ll-keto progesterone as thestarting steroid produced the corre spending '11-keto-17a hydroxyprogesterone.

i ExAMPLE 4 Following the. procedure in Example 1, using 113- ,hydroxyprogeste'rone as the starting steroid produced the eorresponding 11p,17ndihydroxyprogesterone.

' EXAMPLE 5 Followingthe procedures of Examples 1, 2, 3 and 4, the same respective results were obtained upon replacing the nutrient medium of Example 1 by twelve liters of a 'medium'made of'forty grams of glucose, fifteen grams of starch, five grams of ammonium nitrate, three grams of ethylamine, one gram of potassium phosphate (KH POJ, 0.5 gram of magnesium sulfate sulfate O4"4H2 Q 1 am of cupriC Sulfat? 4' H2QIJQQZgram,Qf cobalt itrate I fand 0.01 grarn'l of sodium fluoride (NaF) dilutedtoone rliterwith Kalama oq water, and. ust t @PH 9?. 5.-

.mlExaMrt-a 7 Following the proceduresot Examples 1, 2, 3 and 4,

i the same respective'res ults were obtained'by replacing the nutrientmedium of Example 1 by twelve litersofa medium ma e I tw a ms of n um ate ramo q'ta s m h s ha Z P Q, '0- am/9 potassium chlqride KCl),- 0.5 gram of magnesium sulgramspofigliicose ajnd ten grainsv of corn-steep liquor di- .luted .to one liter wi'thtap water and adjusted to a pH 9M wi hrh sp qri a i V a EXAMPLE 7 yeast autolyzate and ten grams of sucrose diluted to one 1 .lite 'fwith tapwater, produced the corresponding respective resu e In the prfecedirig Examples Into. ,7, inclusive,- the fun{ ;gu s C'ephdldtheciiim fo'seum can be'replaced by strains V I ha thec iun; polybroc'hum or T rich'othe'cium a'rirlt'enbpur'fi fio'x'foibtain1i1flrhydroxylation. LikewiseT Ithe 17,-jhydroxylation illustrated in Examples '1*ito 7', in

i cco rdancewiththe r cedures; Ex 't j ejii q s Y 'b is la h he fqns' sc n Qalqrheciu'mfroselljrg fAT CCf 86851 by Triehotheciimi I rit massage Be un an Re nin (QB-.593] Trichothecium domesticum C.B.S.) 'lrichotheciufri xa'seumlLink (0.3.8.); Trichqthec ium roseum' Link- (with t elusive, is obtained] Table III.

acemes .pseudoverticilliuni form) (C.B.S.'); Trichothecium cyste- A medium was prepared offifty grams of cerelose dextrose, two grams of KH PO 0.5 gram of MgSO 0.3 gram of ZnSO 0.35 gram of K 80 7.5 grams of yeast extract diluted to one liter with tap water and adjusted to a pH of 5.7. Twelve liters of this sterilized medium was inoculated with Trichothecium roseum (Cephalothecium roseum) and incubated aeration for 72 hours. Then six grams of progesterone dissolved in 200 milliliters of ethanol was added to the culture and fermentation with aeration was continued for 48 hours..

Extraction of the beer, containing mycelium, with methylene dichloride gave ten grams of an oily residue, an aliquot of which was separated by paper chromatography to give 11a,17a-dihydroxyprogesterone.

The oily residue was dissolved in 300 milliliters of ethylene dichloride and chromatographed over 480 grams of Florisil synthetic magnesium silicate using 700-milliliter portions of solvent in accordance with Table II. Solid fractions 11 through 14, inclusive, were triturated with ether, the mother liquors were decanted, and the crystalline residues were combined and recrystallized twice from ten milliliters of methanol to yield 426 milligrams of 11a,17a-dihydroxyprogesterone having a melt ing point of 218 to 222 degrees centigrade and an optical rotation [cz] of plus 74 degrees (concentration of 0.98 in chloroform), and characteristic infrared spectrum.

EXAMPLE p Following the process of Example 9, using ll-dehydrocorticosterone in place of progesterone, fermentation and methylene dihcloride extraction gave 7.35 grams of an oily extract. This extract was dissolved in 300 milliliters of ethylene dichloride and chromatographed over 320 grams or" Florisil synthetic magnesium silicate using 490-milliliter portions of solvents in accordance with Solids from fractions 15, 16 and 17 were combined and recrystallized twice from acetone to give 216.5 milligrams of cortisone.

EXAMPLE" '11.

, Followingtheprocess of Example 9, using corticosterone 115,21-dihydroxy-4-pregriene-3,20-dione) ,,,in place of progesterone, produced 17-hydroxycorticosterone (11B,17,2l-trihydioxyr4-pregnene-3,ZO-dione) and cortisone (:,21-dihydroxy-4-pregnene-3,l1,20-trione).

EXAMPLE l2 7 Following the process of Example 9, using 17a,21-

dihydroxy-4-pregnene-3,20rdione,. in place of progesterone, produced 11a,17a,2l-trihydroxy-4-pregncue-3,20- dione.

It is to be understood that the invention is not limited to the exact details of operation or exact species of fungus or compounds shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art and the invention is therefore to be limited only by the scope of the appended claims.

We claim: 7

1. A process for hydroxylating a steroid which includes the introduction of an d-hydroxy group at position 17 of a steroid which comprises growing a species of fungus of the genus Trichothecium under aerobic conditions in an aqueous medium, containing a steroid having a tertiary hydrogen attached to the carbon atom at position 17.

2. A process for the biological introduction of a 17ahydroxyl group into a steroid which comprises growing a species of fungus of the genus Trichothecium under aerobic conditions inan aqueous medium containing a steroid having a tertiary hydrogen attached to the carbon atom at position 17 and recovering the resulting 17ozhydroxylated steroid.

3. A process for the introduction of oxygen into a steroid which comprises growing a species of fungus of the genus Trichothecium under agitated aerobic conditions in a nutrient medium containing a non-steroidal source of assimilable carbon and asteroid having a 17- tertiary hydrogen and having up to and including 22 carbon atoms in the "carbon-to-carbon skeleton andrecovering theresulting 17a-hydroxylated steroid- 4. A process forthe introduction of oxygen into"a steroid which comprises growing a species of fungus .of the genus Trichothecium under agitated aerobic conditions in a nutrient medium containing assimilable phosphate and carbohydrate and asteroid having a 17-tertiary hydrogen and a two carbon atom side chain attached to the 17 position and recovering the resultingljahydroxylated steroid. t 7

A p ocess fo t e n toduct o ofv xy snfinte a steroid which comprises growing a speciesof fungus of the genus Trichothecium under agitated aerobic conditions in a nutrient medium containing assimilablephosphate, non-steroidal carbon and a steroid having a 17- tertiary hydrogen, a two carbon atom side chain attached to the 17-position and a 20-keto. group and recovering the resulting 17a-hydr0xylated steroid.

' 6. A process for the introduction of a l7a-hydroxyl group into asteroid which comprises growing a species of fungus of the genus Trichothecium under agitated aerobic conditions in a nutrient medium containing assimilable phosphate, non-steroidal carbon and a steroid wherein R is selected fromthe groupnconsistingof hydro gen and methyl, R isselected fromthe'group consisting of hydrogen, a-hydroxyl, fl-hydroxyh'and ke'toni c oxygen, and R isselected from the group Consisting of hydrogen,"

hydroxyl and aceyloxy, and recovering the resulting 17a hydroxylated steroid.

7. A process for the introduction of oxygen into a 17-acetyl-17-tertiary hydrogen steroid which comprisesof the genus Trichothecium under aerobic conditions in a nutrient medium containing ll-ketoprogesterone and recovering the resulting l1-keto-l7a-hydrojxyprogesterone.

10. A process for producing l1u,17a-dihydroxyprogesterone which comprises growing a species of fungus of the genus Trichothecium under aerobic conditions in a nutrient medium containing lla-hydroxyprogesterone' and recovering the resulting 11a,17u-dihydroxyprogester- 111A process for producing "11fi,17a'-dihydroxypro-. gesterone which comprises "growing a speciesof fungus of the genus Trichothecium junder, aerobic conditions in,

1 t ,a' nutrient medium"eontaining llfihydroxyprogesterone'* and recovering the resulting 11/8 one.

t'ju-dihydroxyprogesterr covering the resulting l7a-hydr6itylated steroid. 13. A process for the introduction of oxygen into a comprising growing Trichothecium 'rqseum under aerobic aconditions in a nutrient medium containinga steroid hav-, 7 irigja tertiary hydrogen attached to the carbon atom at I positionf17, a two-carbon atom side chain attached to position 17, and a 20-keto group and recovering theresuiting 17a-hydroxylated steroid. t

16. A process for hydroxylating a steroid which includes the introduction .of an u-hydroxy group at position 17 Ma steroid which'comprises growing Trichothecium roseum under aerobic conditions in an aqueous medium, containing a steroid having a tertiary hydrogen :attached to the carbon atom at position 17. I

17. A process for the production of 17a-hYd1'OXY- roseum under aerobic conditions in a nutrient medium containing lld-hydroxyprogesterone and recovering the resulting 11a,17a-dihydroxyprogesterone.,

'rocess for producing gesterone 'which comprises growing Trichothecium roseum vunder aerobic conditions in a nutrient medium containing}lfi-hydroxyprogesterone and recovering the resulting 11B,17u-dihydro ryprogesterone.

- 21.-A process forproducing cortisone-21-ester which .-,c'ornprises growing Trichothecium roseum under aerobic v 5.12., A process for the introduction ofox ygen' intq a w --17-glycolylsteroid which comprises growing a species of j fungus or the genus Trichothecium under agitated aerobic conditions in a nutrient medium containingFassimilable pho'sphate and carbohydrate and. said steroid- ,and"-re- 40,

conditions in a nutrient medium containing -l7-desoxy- "f -'tcorticosterone-2l-ester' and recovering the resulting corti- 1 sone-ZI-ester, I r

. References Cited in theme, of this patent YVUNITED' STATES PATENTS 72*,602169 j Murray et 1. July 18, 1952 17-(21-.acyloxy)-aeetyl-steroid which comprises growing U I agitated aerobic conditions in'anutrient medium containing assimilable phosphate, a carbohydrate and said steroid and recovering. the resulting 17a-hydroxylated steroid. V

14. A process for producing cortisone-Zl-ester which comprises growing a species of fungus of the genus Trichothecium under aerobic conditions in a nutrient medium containing 17-desoxycorticosterone-2l-ester and recovering the resulting cortisone-2l ester. Q

15. A process for producing an oxygenated steroid 'a species of fungus of the genus Trichothecium undei f 2,558,023 Shulluet a1. Nov. 3, 19s

Eppstein et al.:'Vitamins and Hormones, vol. XIV.

- *LOTHER REFERENCES 7. I "Ainsworth. et al.: Dictionary of the Fungi c p 

1. A PROCESS FOR HYDROXYLATING A STEROID WHICH INCLUDES THE INTRODUCTING OF AN A-HYDROXY GROUP AT POSITION 17 OF A STEROID WHICH COMPRISES GROWING A SPECIES OF FUNGUS OF THE GENUS TRICHOTHECIUM UNDER AEROBIC CONDITIONS IN AN AQUEOUS MEDIUM, CONTAINING A STEROID HAVING A TERTIARY HYDROGEN ATTACHED TO THE CARBON ATOM AT POSITION
 17. 